The p53 gene is mutated in over 50 different types of human cancers, including familial and spontaneous cancers, and is believed to be the most commonly mutated gene in human cancer (Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al., Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of mutations in the p53 gene are missense mutations that alter a single amino acid that inactivates p53 function. Aberrant forms of human p53 are associated with poor prognosis, more aggressive tumors, metastasis, and survival rates of less than 5 years (Koshland, Science (1993) 262:1953).
The human p53 protein normally functions as a central integrator of signals arising from different forms of cellular stress, including DNA damage, hypoxia, nucleotide deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8). In response to these signals, p53 protein levels are greatly increased with the result that the accumulated p53 activates pathways of cell cycle arrest or apoptosis depending on the nature and strength of these signals. Indeed, multiple lines of experimental evidence have pointed to a key role for p53 as a tumor suppressor (Levine, Cell (1997) 88:323-331). For example, homozygous p53 xe2x80x9cknockoutxe2x80x9d mice are developmentally normal but exhibit nearly 100% incidence of neoplasia in the first year of life (Donehower et al., Nature (1992) 356:215-221). The biochemical mechanisms and pathways through which p53 functions in normal and cancerous cells are not fully understood, but one clearly important aspect of p53 function is its activity as a gene-specific transcriptional activator. Among the genes with known p53-response elements are several with well-characterized roles in either regulation of the cell cycle or apoptosis, including GADD45, p21/Waf1/Cip1, cyclin G, Bax, IGF-BP3, and MDM2 (Levine, Cell (1997) 88:323-331).
Human p53 is a 393 amino acid phosphoprotein which is divided structurally and functionally into distinct domains joined in the following order from N-terminus to C-terminus of the polypeptide chain: (a) a transcriptional activation domain; (b) a sequence-specific DNA-binding domain; (c) a linker domain; (d) an oligomerization domain; and (e) a basic regulatory domain. Other structural details of the p53 protein are in keeping with its function as a sequence-specific gene activator that responds to a variety of stress signals. For example, the most N-terminal domain of p53 is rich in acidic residues, consistent with structural features of other transcriptional activators (Fields and Jang, Science (1990) 249:1046-49). By contrast, the most C-terminal domain of p53 is rich in basic residues, and has the ability to bind single-stranded DNA, double-stranded DNA ends, and internal deletions loops (Jayaraman and Prives, Cell (1995) 81:1021-1029). The association of the p53 C-terminal basic regulatory domain with these forms of DNA that are generated during DNA repair may trigger conversion of p53 from a latent to an activated state capable of site-specific DNA binding to target genes (Hupp and Lane, Curr. Biol. (1994) 4: 865-875), thereby providing one mechanism to regulate p53 function in response to DNA damage. Importantly, both the N-terminal activation domain and the C-terminal basic regulatory domain of p53 are subject to numerous covalent modifications which correlate with stress-induced signals (Prives, Cell (1998) 95:5-8). For example, the N-terminal activation domain contains residues that are targets for phosphorylation by the DNA-activated protein kinase, the ATM kinase, and the cyclin activated kinase complex. The C-terminal basic regulatory domain contains residues that are targets for phosphorylation by protein kinase-C, cyclin dependent kinase, and casein kinase II, as well as residues that are targets for acetylation by PCAF and p300 acetyl transferases. p53 activity is also modulated by specific non-covalent protein-protein interactions (Ko and Prives, Genes Dev. (1996) 10: 1054-1072). Most notably, the MDM2 protein binds a short, highly conserved protein sequence motif, residues 13-29, in the N-terminal activation domain of p53 (Kussie et al., Science (1996) 274:948-953. As a result of binding p53, MDM2 both represses p53 transcriptional activity and promotes the degradation of p53.
Although several mammalian and vertebrate homologs of the tumor suppressor p53 have been described, only two invertebrate homologs have been identified to date in mollusc and squid. Few lines of evidence, however, have hinted at the existence of a p53 homolog in any other invertebrate species, such as the fruit fly Drosophila. Indeed, numerous direct attempts to isolate a Drosophila p53 gene by either cross-hybridization or PCR have failed to identify a p53-like gene in this species (Soussi et al., Oncogene (1990) 5: 945-952). However, other studies of response to DNA damage in insect cells using nucleic cross-hybridization and antibody cross-reactivity have provided suggestive evidence for existence of p53-, p21-, and MDM2-like genes (Bae et al., Exp Cell Res (1995) 375:105-106; Yakes, 1994, Ph.D. thesis, Wayne State University). Nonetheless, no isolated insect p53 genes or proteins have been reported to date.
Identification of novel p53 orthologues in model organisms such as Drosophila melanogaster and other insect species provides important and useful tools for genetic and molecular study and validation of these molecules as potential pharmaceutical and pesticide targets. The present invention discloses insect p53 genes and proteins from a variety of diverse insect species. In addition, Drosophila homologs of p33 and Rb genes, which are also involved in tumor suppression, are described.
It is an object of the present invention to provide insect p53 nucleic acid and protein sequences that can be used in genetic screening methods to characterize pathways that p53 may be involved in as well as other interacting genetic pathways. It is also an object of the invention to provide methods for screening compounds that interact with p53 such as those that may have utility as therapeutics.
These and other objects are provided by the present invention which concerns the identification and characterization of insect p53 genes and proteins in a variety of insect species. Isolated nucleic acid molecules are provided that comprise nucleic acid sequences encoding p53 polypeptides and derivatives thereof. Vectors and host cells comprising the p53 nucleic acid molecules are also described, as well as metazoan invertebrate organisms (e.g. insects, coelomates and pseudocoelomates) that are genetically modified to express or mis-express a p53 protein.
An important utility of the insect p53 nucleic acids and proteins is that they can be used in screening assays to identify candidate compounds which are potential therapeutics or pesticides that interact with p53 proteins. Such assays typically comprise contacting a p53 polypeptide with one or more candidate molecules, and detecting any interaction between the candidate compound and the p53 polypeptide. The assays may comprise adding the candidate molecules to cultures of cells genetically engineered to express p53 proteins, or alternatively, administering the candidate compound to a metazoan invertebrate organism genetically engineered to express p53 protein.
The genetically engineered metazoan invertebrate animals of the invention can also be used in methods for studying p53 activity, or for validating therapeutic or pesticidal strategies based on manipulation of the p53 pathway. These methods typically involve detecting the phenotype caused by the expression or mis-expression of the p53 protein. The methods may additionally comprise observing a second animal that has the same genetic modification as the first animal and, additionally has a mutation in a gene of interest. Any difference between the phenotypes of the two animals identifies the gene of interest as capable of modifying the function of the gene encoding the p53 protein.